Trypanosomes are parasitic protozoan hemoflagellates that cause health problems in developing countries. These organisms diverged from other eukaryotes early in evolution and possess many unique RNA processing pathways such as uridine insertion/deletion editing of mitochondrial mRNAs. Studies of the RNA editing and guide RNA maturation processes emphasize RNA uridylylation reactions as crucial for RNA biogenesis in mitochondria of Trypanosoma brucei. We discovered three Terminal Uridylyl Transferases (TUTases), enzymes of unique structures and essential functions. This proposal focuses on: 1) functions of the RET1-catalyzed 3' -uridylylation in processing RNA precursors; 2) the mechanism by which RET2 guides U-insertion; and 3) the biological role of MEAT1. We consider this research to be indispensable for the development of TUTase inhibitors as potential trypanocides. The Specific Aims are: 1. Investigate functions of RET1-catalyzed 3' -uridylylation of small and ribosomal RNAs. The editing is directed by trans-acting guide RNAs which are post-transcriptionally modified by the 3' U -addition. Similar U-tails are also found in ubiquitous gRNA-like molecules and in rRNAs. We propose that uridylylation stabilizes gRNA-like molecules, which direct nucleolytic cleavage of maxicircle- and minicircle-encoded multicistronic transcripts. We will analyze functions, sequence diversity, and stability of short RNAs by next-generation sequencing and biochemical methods. 2. Determine the mechanism of the RET2-mediated U-insertion editing reaction. We propose that the fidelity of the U-insertion editing is determined by RET2's intrinsic selectivity for UTP and RNA substrates while complex association facilitates the editing efficiency. Structure-based predictions will be tested by a novel RNAi-based inducible genetic knock-in system. 3. Establish the function of MEAT1 TUTase. MEAT1 is an exclusively U-specific TUTase which associates with a 20S editosome-like particle and is essential for the parasite's viability. We propose to investigate whether U-insertion editing is accomplished by distinct RET2- and MEAT1-dependent mechanisms. MEAT1-specific U-insertion editing sites and interacting partners will be identified by in vivo crosslinking and quantitative mass spectrometry.